In animals with internal fertilization, seminal proteins transferred with sperm play crucial roles in the fertility of both males and females. This is best understood in the model system Drosophila, whose seminal proteins ("Acps") induce females to increase egg production, store sperm, and modify their behavior, but also to die younger than unmated females. We have identified 90% of all Acps in the fly genome (52 genes). They include members of conserved seminal-protein classes such as peptides, sperm-binding proteins and protease inhibitors; 29% show signs of having evolved rapidly, a trait seen for reproductive proteins in many organisms. We have also identified effectors and genes regulated by Acps in mated females. Our goal is to determine the Acps that mediate specific reproductive changes in females, and how they carry out their functions. In Aim 1we will screen for the reproductive function of individual Acps. We will assess the targeting, biochemical activity and structure of these molecules. We will determine their in vivo functions by ectopic expression assays in females, and by the consequences of removing individual Acps from males. We have adapted the Gateway cloning system to carry out these assays with high efficiency. In Aim 2 we will analyze in detail the functions of individual Acps that regulate egg production, sperm storage or lifespan effects/protease inhibition. Initially we will extend our analysis of three Acps, each representing one of these areas. We will determine molecular requirements for their activity, the downstream genes/effectors they regulate, and their binding proteins. We will then similarly analyze Acps identified in Aim 1 to be important in egg production, sperm storage or proteolysis regulation. Through this revised research plan we will obtain a comprehensive functional view of the fly tissue analogous to the mammalian prostate gland, and a specific view of the actions of individual seminal proteins and the responses they trigger in females. This information will help us understand the molecular interactions between males and females, and to evaluate hypotheses about the functions and evolution of reproductive proteins. In addition to their relevance to reproductive mechanisms in a range of organisms including mammals, we anticipate that our findings will have practical applications in the control of insect vectors of human disease.